The invention is concerned with a method for the production of activated marked tumor-specific T cells by co-cultivating tumor cells from a patient with T cells from that patient, a therapeutic composition containing such activated T cells as well as the use thereof in tumor therapy.
Tumor-specific T lymphocytes recognize peptides derived from proteins synthesized by tumor cells and presented on their cell surface by MHC molecules (Lurquin et al., Cell 58 (1989) 293 and Hellstrxc3x6m, K. E., et al., The Biologic Therapy of Cancer, J. B. Lippincot Co., Philadelphia (1991) p. 35). However, T cells require two activating signals to express full effector functions (Mueller, D. L., et al., Annu. Rev. Immunol. 7 (1989) 445). Signal 1 is generated when the T cell receptor (TCR) interacts with the MHC peptide complex. Signal 2 is provided by costimulatory molecules expressed by professional antigen-presenting cells (APC). Many tumors, particularly those of non-hematopoietic origin, do not express costimulatory molecules and thus fail to activate tumor-specific T lymphocytes (Chen, L., et al., Immunol. Today 14 (1993) 483). This finding has provided a rationale for the introduction of genes encoding costimulatory molecules into tumor cells to increase their immunogenicity and vaccination potential.
Among the different costimulatory molecules, B7 proteins (e.g. B7-1, B7-2 and B7-3) are of particular interest since they are expressed on professional APC (Vandenberghe, P., et al., Int. Immunol. 3 (1993) 229; Guinan, E. C., et al., Blood 84 (1994) 3261-3282; WO 95/03408). These costimulatory molecules interact with CD28 and CTLA4 counter-receptors expressed on most T cells leading to a marked increase of IL-2 production, proliferation and acquisition of effector function in both CD4+ and CD8+ T cells (Azuma, M., et al., J. Immunol. 115 (1993) 2091). Blocking the ligation of B7 with a soluble CTLA4-Ig chimeric molecule provokes unresponsiveness in vitro, which has dramatic suppressive effects on the humoral response and graft rejection in vivo. In addition, it has been shown that the transfection of the B7-1 gene into different mouse tumor lines can lead, in some cases, to both their primary rejection and the establishment of a protective immunity (Chen, L., et al., J. Exp. Med. 179 (1994) 523 and Ramarathinam, L., et al., J. Exp. Med. 179 (1994) 1205). However, these studies have revealed a limited efficiency of B7-1 activity on T cell-dependent tumor immunity.
The efficiency of B7 costimulation of anti-tumor T cells is enhanced by cooperation between B7 and ICAM-1, whereby an efficient tumor-specific immune response is stimulated. This effect is dependent on the recruitment of a potent inflammatory reaction (Cavallo, F., et al., Eur. J. Immunol. 25 (1995) 1154-1162).
Molecules of the B7 family are CD28 counter-receptors expressed on APCs. B7-1 was characterized and sequenced in Freeman, G. J., et al., J. Immunol. 143 (1989) 2714-2722. B7-2 and B7-3 were characterized and sequenced in Freeman, G. J., Science 262 (1993) 909-911 and WO 95/03408. The B7 molecules are members of the Ig supergene family with two Ig-like domains (IgV and IgC) and a transmembrane domain. It is suggested that the B7 molecules exist as a monomer or a homodimer on the cell surface, but little, if any, evidence suggests that it can form a heterodimer with CD28 (Lindsten, T., et al., J. Immunol. 151 (1993) 3489). The B7 molecules have a higher affinity for CTLA-4 than for CD28. The genes of the B7-1 and B7-2 molecules have been localized to chromosomal region 3q13.3-3q21. Though these molecules were not highly homologous at the DNA level, they share the identical Ig supergene family structure and the ability to bind to CD28 and CTLA-4, as mentioned above.
However, it was found that B7-1 and B7-2 differ in their appearance after B cell activation. B7-2 appears on the cell surface within 24 hours of B cell activation and B7-1 appears later (Boussiotis, V. A., et al., Proc. Natl. Acad. Sci. USA 19 (1993) 11059). It was further found that in unstimulated human monocytes B7-2 is constitutively expressed whereas B7-1 expression is induced after activation (Azuma, M., et al., Nature 366 (1993) 76). B7-3 is also described in Boussiotis et al. B7-3 has not yet been molecularly cloned.
In WO 95/03408 it is suggested to modify a tumor cell to express B7-2 and/or B7-3 by a transfection of the tumor cell with the nucleic acid encoding B7 in a form suitable for expression of B7 on the tumor cell surface. Alternatively, the tumor cell is modified by contact with an agent which induces or increases the expression of B7 on the tumor cell surface. It is further suggested to couple B7-2 and/or B7-3 to the surface of the tumor cell to produce a modified tumor cell. The term xe2x80x9ccouplingxe2x80x9d as used in WO 95/03408 refers to a chemical, enzymatic or other means (e.g. antibody) by which B7-2 and/or B7-3 is linked to a tumor cell such that the costimulatory molecule (B7) is present on the surface of the tumor cell and is capable of triggering a costimulatory signal in T cells. It is further suggested to cross-link B7 chemically to the tumor surface, using commercially available cross-linking reagents. Another approach would be to couple B7-2 and/or B7-3 to a tumor cell by a B7-specific antibody which binds to both the costimulatory molecule B7 and a cell surface molecule on the tumor cell.
The production of activated tumor-specific T cells may be accomplished by co-cultivating tumor cells from a patient, which tumor cells carry, on their surface, such a costimulatory molecule, with T cells from that patient. Modifying such tumor cells with B7 according to the known method involves a number of drawbacks, however, and is rather unsuitable for routine therapy. Transfecting the tumor cells with the nucleic acid encoding a costimulatory molecule usually is not very effective. In addition to this, it is necessary that the transfected and non-transfected cells should be separated, in a laborious procedure, prior to co-cultivation with the activated T cells. McHugh, R. S., et al., Proc. Natl. Acad. Sci. USA 92 (1995) 8059-8063 suggest to introduce B7-1 onto the surface of tumor cells by using a purified GPI (glycosyl-phosphatidyl-innositol) anchored B7-1 molecule (GPI-B-7) which is able to bind its cognate ligand CD28 and incorporate itself into tumor cell membranes after a short incubation. However, the stability of the GPI-B-7 on the surfaces of irradiated tumor cells is limited and the cells do retain only minimal presentation of B7 capable of effective binding to CD28.
Coupling of B7 to a tumor cell by using a B7-specific antibody which binds both the costimulatory molecule and the cell surface molecule of the tumor has also severe disadvantages. B7 antibodies which are described in the state of the art bind to B7 unfortunately in such a way that the binding of B7 to CD28 decreases dramatically or is completely inhibited. The reason for this is that all known anti-B7-1 and anti-B7-2 monoclonal antibodies interact with CD28 and thus inhibit the T cell response (Azuma, M., et al., J. Exp. Med. 175 (1992) 353-360; Azuma, M., et al., J. Immunol. 149 (1992) 1115; Azuma, M., et al., J. Exp. Med. 177 (1993) 845; Caux, C., et al., J. Exp. Med. 180 (1995) 1841-1847).
It is therefore the object of the present invention to provide a method for the production of activated tumor-specific T cells which can be carried out in a simple manner and exhibits a high efficacy.
The subject-matter of the invention is a method for the production of activated tumor-specific T cells by co-cultivating, ex vivo, tumor cells from a patient with T cells from that patient, comprising the steps of
i) incubating the tumor cells with a first fusion protein obtained from a B7 protein and one partner of a biological binding pair and a second fusion protein obtained from an antibody against a cell surface antigen and the other partner of the biological binding pair;
ii) inhibiting the proliferation of the tumor cells prior to or after that incubation;
iii) co-cultivating the tumor cells with the T cells to be activated, until activation of the T cells is attained;
iv) separating the activated T cells from the tumor cells.
T cells of a patient are isolated from peripheral blood lymphocytes (PBMC), which have been prepared from buffy coat of normal human blood samples (Dellabona, P., et al., J. Exp. Med. 177 (1993) 1763-1771). After centrifugation, the mononucleic cells are collected and propagated (Dellabona, P., et al., J. Exp. Med. 177 (1993) 1763-1771). From this preparation, CD4+ and/or CD8+ lymphocytes can be isolated by means of magnetic activated cell sorting (MACS).
The T cells which are used for activation can be generated from a patient according to known methods, preferably by a simple passage of PBMC on any long wool column, whereby B cells and monocytes are excluded (Julius, M. H., et al., Eur. J. Immunol. 3 (1973) 645). CD8+ (with or without CD4+) T cells are purified from the peripheral blood of the patient in vitro by a sorting method, preferably by immunomagnetic sorting. In addition, it is preferred to use a mixed population of tumor-infiltrating T cells (TIL) and purified CD8+ T cells which are obtained from a surgical tumor specimen according to Anichini, A., et al., J. Exp. Med. 177 (1993) 989.
The phrase xe2x80x9cactivated tumor-specific T cellsxe2x80x9d preferably denotes tumor-specific T cells which are capable of killing in a specific and restricted manner the tumor cells originally used to activate them. The activation is MHC restricted in the sense of Townsend, A., and Bodmer, H., Ann. Rev. Immunol. 7 (1989) 601.
Generation of tumor specific T cells either total PBMC, or purified CD8+ T cells are cultured in 24-well plates at a ratio of 10.1 to 5.1 with non-replicatig tumor cells (irradiated or mit. C treated or both) which are either autologous or semi-allogeneic, in 2 ml of standard RPMI medium containing 5% human serum, at 37xc2x0 C. Multiple cultures containing 2 to 5 millions of PBMCs or 1 to 2 millions of CD8+ T cells can be set. The tumor cells have been prepulsed with a saturating concentration (to be determined depending on the different kind of constructs) of soluble B7-1 or B7-2 Igxc3x97antitumor mAb. Recombinant human IL-2 is added to the culture at 5 U/ml at day +5 of the culture and maintained until day +10, after which its concentration is raised to 10 U/ml. At day +15 of the culture, living T cells are recovered from the cultures by a centrifugation over a Ficoll gradient and re-stimulated using the same non-replicating tumor cells prepulsed with the recombinant B7-1 or B7-2 Igxc3x97anti-tumor mAb at a ratio T cell/tumor of 2:1, and a concentration of one million T cells per ml. This restimulation step is performed in 24-well plates, in standard RPMI medium containing human serum supplemented with 2 U/ml recombinant human IL-2. At day +5, the concentration of rhIL-2 is raised to 10 U/ml and maintained as such until day +15.
The T cells can be restimulated a third time as described above, before being tested in a conventional cytotoxicity test (Lanzavecchia, A., Nature 319 (1986) 765-767) against the tumor cells used for re-stimulation in vitro, and unrelated tumor cells for control. The specificity of the T cell line for the tumor is judged according to the level of cytotoxicity shown in the assay. A specific killing activity of about 30-40% can be considered relevant for therapeutic interest. In this case, the tumor specific polyclonal T cell line can be expanded further using a polyclonal activator: PHA in the presence of irradiated allogeneic feeder cells (allogeneic PBMC) and 10 U/ml of rhIL-2; or anti-CD3 mAb+B7-1 IgM and 10 U/ml rhIL-2. At day +15 of restimulation, rhIL-2 is raised to 20 U/ml for 5 days and then to 50 U/ml for another 5 days. By different cycles of restimulation it is possible to reach the desired number of activated T cells to be reinfused in the patient.
In a preferred embodiment, the proliferation of the tumor cells is inhibited prior to or after the incubation according to step i). This may be accomplished, for instance, by means of irradiation or by use of mitomycin C. For irradiation, preferably 3000-5000 Rad are used for inhibition with mitomycin C. preferably 50-100 xcexcg/million of cells are used. Mitomycin C is preferred for inhibition because it prolongs the survival of tumor cells during re-stimulation of T cells.
In a further preferred embodiment of the invention, the activated T cells are marked, preferably after activation. Such a market preferably is a molecule which is presented on the surface of the marked cell. It is therefore particularly preferred to transform the T cells with the nucleic acid which codes for a protein which is presented on the surface of said cell. Such cell surface proteins or antigens are, for example, CD24 (J. Cell. Biochemistry Supplement 17E, page 203, abstract S210), LDL or NGF receptor (WO 95/06723).
After autologous transplantation of said activated T cells which are marked with such a gene product it is possible to trace these cells directly after transplantation in the patient. This gene marking will allow to monitor and compare the efficiency of the therapy with activated tumor-specific T cells.
In a preferred embodiment of the invention, the T cells can further be transformed with a suicide gene. Such a gene causes directly or by mediators the death of the infected cell (cf. WO 92/08796 and PCT/EP94/01573) for in vivo-specific elimination of these cells after successful therapeutic treatment. For this purpose, there is preferably applied the thymidine kinase gene, which confers to the transduced activated T cells in vivo sensitivity to the drug Gancyclovir for in vivo-specific elimination of cells. If, for example, the patient develops signs of an acute incompatibility of the activated T cells, for example like a graft versus host disease (GVHD), with increasing liver function enzymes and a positive skin biopsy, it is preferred to administer i.v. two doses of about 10 mg/kg of the drug Gancyclovir. This results in a reduction of marked activated T cells without a considerable reduction of other lymphocytes.
The diphtheria toxin gene is also preferred as a suicide gene, which is described in WO 92/05262. For the in vivo-specific elimination of the activated T cells, it is also possible to induce a cell apoptosis. It is thus preferred to use a modified FAS receptor and a dose of a related ligand.
The tumor cells of the patient are taken from a surgical specimen. An aliquot of tumor cells can be used to regenerate a tumor cell line either in vitro or in vivo in immunodeficient mice, for subsequent stimulation of T cells. The rest of the fresh tumor cells are used for the direct stimulation of the T cells. Such tumor cells are, for example, melanoma, carcinoma (e.g. breast, cervix, head and neck, colon, lung, kidney, stomach), sarcoma, lymphoma or leukemia.
The term xe2x80x9cbiological binding pairxe2x80x9d is understood to mean a combination of two molecules which have a high specific binding capacity with respect to one another. Such binding pairs are, for example, biotin/avidin (or streptavidin/neutravidin), or sugar/concanavalin A. Preferably, the affinity constant of the binding is kd less than 1 nmol/l. Preferably, higher affinity constants are applied. For this reason, the biotin/avidin or streptavidin interaction is preferred because of their high affinity. This interaction is stronger than any other receptor ligand in a body. Therefore, it can be used in vivo to conjugate two components of a bifunctional reagent. Methods of biotinylatin are described in Harlow, E., and Lane, D., Antibodies, Cold Spring Harbor Laboratory (1988) 341. Biotinylation or cross-linking with avidin or streptavidin or neutravidin is carried out according to the methods well-known to one skilled in the art.
A preferred technique for pairing two protein molecules is chemical crosslinking, which forms a stable covalent bridge. Many bifunctional crosslinkers are commercially available. There is particularly preferred SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate) crosslinker.
As B7 protein, part of all of B7, preferably of B7-1, B7-2 or B7-3 can be used. Preferably, there are used parts of B7 proteins which include at least the N-terminal variable domain. There are particularly preferred B7-1 or B7-2 proteins and derivatives as described in WO 95/03408, which is for this matter incorporated herein by reference. Since the binding of the B7 fusion protein to the cell surface is effected via the interaction between the biological binding pair and the binding of the anti-surface antigen-antibody, it is not necessary that the B7 molecule should still contain the transmembrane domain.
The second fusion protein contains an antibody against a cell surface antigen and the other partner of the biological binding pair. As antibodies, antibodies that are directed against a great number of cell surface antigens can be used. Those cell surface antigens need not necessarily be tumor cell-specific surface antigens. For this reason, it is preferred to use antibodies against surface antigens which occur to a large extent on cell surfaces, such as ERB B2 or the transferring receptor. It is also possible, however, to use antibodies against suitable tumor-associated antigens like CEA for colon carcinomas, lung carcinomas, mammary carcinomas, CD33 for myeloid leukemias; CD19/CD20 CALLA and CD38 for B cell leukemias, lymphomas, myelomas; Met for gastric carcinomas; OVCA (MOV-18) for ovarian carcinomas; or melanoma-specific antigens.
Preferably, co-cultivation of the activated tumor cells with the T cells is carried out in the presence of small doses of lymphokines (such as IL-2, IL-6, IL-7) which, in addition, are capable of stimulating T cells well. Several rounds of re-stimulation are preferred in order to expand to large numbers tumor-specific effector T cells. Three to four days after the last re-stimulation in vitro, the tumor effector T cells are reinnoculated i.v. into the patient. The number of T cells that must be transferred into the patient is variable and can be found out according to established protocols. Preferably, one i.v. infusion for four weeks, consisting of 107-109 cells/sq.m of body surface area, is used. Established protocols are described, for example, in Greenberg, P. D., Adv. Immunol. 49 (1991) 281-355, and Riddel, R. R., et al., Science 257 (1992) 238-241. At the time of adoptive transfer, patients can be vaccinated with immunogenic tumor cells as well as treated with soluble recombinant tumor-specific B cell molecules to provide further re-stimulation in vivo. This can be achieved by injecting i.v. in the patients a predetermined amount of the soluble B7 conjugate, whereby the mAb binds to the surface of the cells of the tumor diagnosed. If the tumor expresses more than one suitable marker, more than one B7xc3x97anti-tumor mAb molecule can be injected into the patient. The amount of soluble reagents and the schedule of injections are determined during the preclinical and clinical trails using, for instance, radio-labeled proteins to monitor its clearance and the efficiency of targeting into the tumor mass. All the essential parameters for this kind of treatment must be derived from the conventional experience of pharmacological and nuclear medicine and are not difficult to find out. It will also be essential to monitor the neutralizing antibody response that the patients can mount against the recombinant proteins.
As mentioned above, it is preferred to monitor the activated T cells in vivo after application. In this case, the adoptive immunotherapy is based on ex vivo expansion of tumor-specific T cells, which are marked preferably with LNGFR and reinnoculum into a patient, preferably together with the tumor-specific soluble B7 conjugate. In this case, the soluble B7 conjugate will re-stimulate transferred tumor-specific effector T cells at the site of the residual tumor mass, allowing the optimal amplification in vivo of the immune response. In fact, in the absence of soluble B7 conjugates (or in the absence of another adequate costimulation), the transferred antitumor T cell blast may perform fewer cycles of killing, after which they can be functionally inactivated or physically eliminated by programmed cell death. The marking of the tumor-specific T cells transferred during the adoptive immunotherapy may allow to monitor the efficiency of this approach by determining the persistence in the patient of transferred T cells.
Also within the scope of the scope of the invention is a kit for treating a tumor in a patient, wherein the kit comprises a fused protein. The fused protein comprises a first fusion protein and a second fusion protein linked by the two partners of the biological binding pair defined above bound together. The first fusion protein comprises an antibody against a cell surface antigen of the patient or at least two Fab fragments of the antibody and one partner of a biological pair. The second fusion protein comprises the other partner of the biological binding pair and a B7 protein. The fused protein can be prepared by incubating the first and second fusion proteins together to form the fused protein, followed by isolation of the fused protein using a method known in the art for separating biomolecules based on a difference in size. Examples of the isolation method include ultrafiltration, chromatography and electrophoresis.
In a preferred embodiment, the kit further comprises an inhibitor of tumor cell proliferation and/or at least one lymphokine. Preferred lymphokines are IL-2, IL-6 and IL-7. The xe2x80x9cinhibitor of tumor cell proliferationxe2x80x9d is a substance which is capable of inhibiting the proliferation of tumor cells. Examples of the inhibitor of tumor cell proliferation are anti-tumor drugs, such as alkylating agents (e.g. mechlorethamine), antimetabolites, folic acid analogs (e.g. methotrexate), pyrimidne analogs (e.g. 5-fluorouracil), vinca alkaloids (e.g. vinblastine), epipodophyllotixins, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin C, L-asparaginase, interferon, cisplatin, carboplatin, mitoxanthrone, hydroxyurea, procarbazine, mitotane, aminoglutethimide, prednisone, hydroxyprogesterone, diethystibestrol, tamoxifen, testosterone proprionate, flutamide, and leuprolide.
Also within the scope of the invention is a kit for treating a tumor in a patient, wherein the kit comprises a fused protein. The fused protein comprises a first fusion protein and a second fusion protein linked via a linking group. The linking group comprises a partner B of a biological binding pair as defined above, with the two partners of the biological binding pair referred to as partner A and partner B in this embodiment. In other words, the fused protein comprises a first fusion protein, partner B, and a second fusion protein. The first fusion protein comprises an antibody against a cell surface antigen of the cells of the tumor to be treated or at least two Fab fragments of said antibody and partner A. The second fusion protein comprises partner A and a B7 protein. Examples of partner A and partner B are (I) biotin ad avidin, streptavidin or neutravidin or (II) sugar and concanavalin A. Preferably, partner A is biotin and partner B is avidin, streptavidin or neutravidin. The B7 protein can be B7-1 or B7-2. The fused protein can be prepared by incubating the first fusion protein, partner B and the second fusion protein together, followed by isolation of the fused protein based on the size of the fused protein using a known method, such as ultrafiltration, chromatography or electrophoresis. In a preferred embodiment, the kit further comprises an inhibitor of tumor cell proliferation and/or at least one lymphokine. Preferred lymphokines are IL-2, IL-6 and IL-7. The xe2x80x9cinhibitor of tumor cell proliferationxe2x80x9d is a substance which is capable of inhibiting the proliferation of tumor cells. Examples of the inhibitor of tumor cell proliferation are anti-tumor drugs disclosed above.
Also within the scope of the invention is a method of using one of the above kits in treating a tumor of a patient by administering an effective amount of the fused protein in the kit to the patient. The above kits can be used in a method to treat a tumor in a patient by targeting tumor cells from the patient. The method comprises obtaining T cells and cells from the tumor to be treated from the patient. The proliferation of the tumor cells is inhibited and the tumor cells are incubated with the fused protein of the kit to obtain targeted tumor cells. Preferably, the proliferation of the tumor cells is inhibited before or after the incubation with the fused protein. The T cells are then co-cultivated with the targeted tumor cells to activate the T cells. The activated T cells are separated from the targeted tumor cells to obtain activated tumor-specific T cells, which are administered to the patient to stimulate immunity of the patient against the tumor in order to treat the tumor. Preferably, the activated tumor-specific T cells are administered via intravenous infusion. The amount of activated tumor-specific T cells to be administered would depend on the type of tumor to be treated and the physical condition of the patient. One skilled in the art would know how to adjust the amount to be administered. The preferred amount of activated tumor-specific T cells to be administered is about 107 to about 109 activated tumor-specific T cells per square metet of body surface area of the patient.
Another aspect of the invention are activated tumor-specific T cells. Said activated tumor-specific T cells are activated by tumor cells targeted with a B7 protein. The targeted tumor cells have a B7 protein stably attached to the surface of the tumor cells. The activated tumor-specific T cells are prepared by one of the above disclosed methods. The activated tumor-specific T cells can be used by treating the tumor after administration of an effective amount of the T cells to the patient. The prefered amount of activated tumor-specific T cells to be administered is about 107 to about 109 activated tumor-specific T cells per square metet or body surface area of the patient.